Method For Bacterial Detection Using Film Formation Promotion With Enhanced Corrosion Imbalance

ABSTRACT

A system and method adapted to have high sensitivity to the formation of biofilm by mixed community bacteria in fluids. The system enhances corrosion imbalance by differentiating conditions between metal sensor elements immersed in the liquid being monitored. The liquid may be diverted to and flowed through a sample chamber where adjacent sensor elements may reside in different flow velocity or temperature regions. The differentiated conditions allow for different film formation on one of the sensor elements relative to the other, and also more quickly than in the main system from which the sampled liquid has been diverted. The differentiated formation allows the use of measurement of polarization current between the metal sensors to produce data with superior resolution relative to prior methods. The speed of film formation promoted by the differentiated conditions allows for determination of risk of film formation in the main system prior to that film&#39;s formation in the main system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/656,729 to Howland, filed Jul. 21, 2017, which claims priority toU.S. Provisional Application No. 62/483,570 to Howland, filed Apr. 10,2017, which are hereby incorporated by reference in their entirety.

BACKGROUND Field of the Invention

This invention relates to the early detection of live water bornebiology.

Description of Related Art

In industrial, domestic, and medical aquatic systems there are manyfactors which can create problems by reducing the efficiency of theprocess concerned or by creating toxicity problems. For example, waterused in most of these systems will usually contain some form of livingmicroorganisms which under the conditions in the systems can multiplyand cause a number of problems. Initially the microbes may be suspendedin the water. These so-called planktonic microorganisms are relativelyeasy to kill using conventional biocides. Although they can causeproblems, for instance if the microorganisms are pathogenic, in generalthey do not significantly affect the overall efficiency of the system.

The planktonic microorganisms can however become adherent to internalsurfaces in the system when they are known as “sessile”. Sessilebacteria can proliferate and some bacteria generate a slime composedmainly of polysaccharide and form a film upon the surface. These filmscontribute to inefficiencies in the systems for various reasons. Forinstance, the films will reduce efficiency of conductive heat transferthrough the surfaces and, since they are highly elastic, increase fluidfrictional resistance at the surface dramatically. Furthermore sometypes of bacteria produce compounds which may be environmentallyhazardous or may lead to corrosion of metal in the surface to which thefilm is attached. Sulphate reducing bacteria or SRB's in particular cangive rise to serious corrosion of metal surfaces.

Bacteria in biofilms are in general found to be difficult to get rid of,partly because chemical biocides must penetrate the slime before theyreach the target microorganisms deep within the films. Because of theproblems that biofilms can create, a dose of a suitable biocide may beperiodically necessary to prevent development of films.

The microbial cells growing in biofilm may be physiologically distinctfrom planktonic cells of the same organism. When a cell switches fromplanktonic to a biofilm mode of growth, it may undergo a phenotypicshift in behavior in which large suites of genes are differentiallyregulated.

What is needed is a system and method for determining whether a fluidsystem, such as a water delivery system, has reached a threshold levelof planktonic bacteria of a type or types which will form a biofilm.What is also needed is a system which can determine whether suchmicrobes are present in advance of the formation of a biofilm whichinterferes with system function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a system for bacteria detection using film formationpromotion according to some embodiments of the present invention.

FIG. 1B is a system for bacteria detection using film formationpromotion according to some embodiments of the present invention.

FIG. 2 is a view of a sample flow chamber according to some embodimentsof the present invention.

FIG. 3 is a view of a sample flow chamber according to some embodimentsof the present invention.

FIG. 4 is a view of a sample flow chamber with some film formationaccording to some embodiments of the present invention.

FIG. 5 is a view of a sample flow chamber with some film formationaccording to some embodiments of the present invention.

FIG. 6 is a view of a sample flow chamber with some film formationaccording to some embodiments of the present invention.

FIG. 7 is a view of is a view of a sample flow chamber with some filmformation according to some embodiments of the present invention.

FIG. 8 is a view of is a view of a sample flow chamber with some filmformation according to some embodiments of the present invention.

FIG. 9 is a view of a test system with two sample flow chambersaccording to some embodiments of the present invention.

FIG. 10 is view of a sample flow chamber with two sets of test probesaccording to some embodiments of the present invention.

FIG. 11 is view of a sample flow chamber with two sets of test probesaccording to some embodiments of the present invention.

FIG. 12 is view of a sample flow chamber with two sets of test probesand some film formation according to some embodiments of the presentinvention.

FIG. 13 is an illustrative sketch of microbial populations on metalsubstrates.

FIG. 14 is a system for bacteria detection using film formationpromotion according to some embodiments of the present invention.

FIG. 15 is a system for bacteria detection using film formationpromotion according to some embodiments of the present invention.

FIG. 16A is a system for bacteria detection using film formationpromotion with a flow restriction device according to some embodimentsof the present invention.

FIG. 16B is a flow restriction device according to some embodiments ofthe present invention.

FIG. 17 is a system for bacteria detection using film formationpromotion according to some embodiments of the present invention.

SUMMARY

A system and method adapted to have high sensitivity to the formation ofbiofilm by mixed community bacteria in fluids. The system enhancescorrosion imbalance by differentiating conditions between metal sensorelements immersed in the liquid being monitored. The liquid may bediverted to and flowed through a sample chamber where adjacent sensorelements may reside in different flow velocity or temperature regions.The differentiated conditions allow for differential film formation onone of the sensor elements relative to the other, and also more quicklythan in the main system from which the sampled liquid has been diverted.The differentiated formation allows the use of measurement ofpolarization current between the metal sensors to produce data withsuperior resolution relative to prior methods. The speed of filmformation promoted by the differentiated conditions allows fordetermination of risk of film formation in the main system prior to thatfilm's formation in the main system.

DETAILED DESCRIPTION

Groups of otherwise unrelated organisms, which may all be bacteria, mayinteract by a mechanism of nutrient succession that ensures bothcooperation between species and maximum utilization of the primarynutrients. Such groups inevitably have a structural component and theyexist as aggregates in the form of flocculents or biofilms. Diffusionalresistance to oxygen penetration, coupled with its uptake by aerobicspecies, can result in the creation of anoxic and reducedmicroenvironments within the central regions of the consortium ofspecies. A general model for a microbial consortium would therefore be anutritionally linked group of aerobic, facultative, and anaerobicbacterial species displaying heterotrophic hydrolytic and fermentativeactivity, acetogenesis, and a terminal oxidative stage involving eithermethanogenesis or sulphate reduction. In a biofilm, particular organismsexist within appropriate microenvironments which are created by theirown and other organisms' activities and are maintained by virtue of thepolymeric matrix within which they are imbedded.

Biofilms are dynamic structures. The pattern of their development can bedivided into phases of transport of molecules and cells to thesubstrate, attachment, increase in biomass (cells and extracellularpolymeric substances), and detachment. In a multispecies biofilm thissituation may be complicated by the requirement for organism A toestablish the necessary conditions, for example anaerobiosis, beforeorganism B can become established within the biofilm.

As the understanding of the nature of biological corrosion mechanismshas increased, the terms “biocorrosion” and “microbial corrosion” havebeen replaced by the term “microbially influenced corrosion”. This is areflection of there being a range of quite different microbial processesinvolved, and the fact that they operate primarily by stimulatingpre-existing electrochemical mechanisms of corrosion rather than byintroducing any novel reaction schemes of their own.

The main instances of microbially influenced corrosion resulting fromthe action of sulphate reducing bacteria are noted with cast iron andmild steel. In the case of buried cast iron pipes, the effect can bedramatic, with the complete removal of the iron leaving behind anapparently unaltered structure which is composed solely of graphite andretains none of the strength of the original material. It is with mildsteels, however, that the sulphate reducing bacteria have their majorimpact.

With the dramatic and damaging impacts the formation of biofilms canhave on an operating system it is of vital importance that live bacteriaof types which may form a biofilm together be detected prior to theformation of biofilm in the operating system. In embodiments of thepresent invention, the presence of live film forming planktonic bacteriais detected using a system which promotes microbially influencedcorrosion and also film formation in a test chamber at an acceleratedrate. This accelerated film formation accentuates differential filmgrowth on sensor tips such that film growth can be detected veryquickly. The detection of this differential film growth alerts thesystem operator that live film forming bacteria are present in fluid ofthe main operating system, and may raise an alert prior to film havingformed in the main operating system. The system operator may then usebiocide or other means to prevent film formation in the main operatingsystem.

In some embodiments of the present invention, as seen in FIG. 1A, asystem for bacteria detection using film formation promotion iscomprised of a test device 100 coupled to and adapted to route fluidfrom a fluid system, such as a water system, via an inlet tube 101. Theinlet tube 101 routes fluid into the chamber inlet 108 of a sample flowchamber 106. In some aspects, fluid from the fluid system is supplied tothe inlet tube 101 using a pump which pumps water from the main fluidsystem. In some aspects, fluid from the fluid system is supplied to theinlet tube 101 via gravity. In some aspects, fluid from the fluid systemis metered using a metering pump. In some aspects, the test device 100is inverted to utilize gravity to create flow in the sample flow chamber106. In some aspects, a supply pump which pumps water from the mainfluid system into the inlet tube 101 is utilized to create flow in thesample flow chamber 106.

In some embodiments, a probe apparatus 103 is removably inserted into anopening 110 in the sample flow chamber 106. The probe apparatus 103 mayhave a first test probe 104 and a second test probe 105 coupled to amounting unit adapted to inserted into the opening in the sample flowchamber 106. In some embodiments, as seen in FIG. 1A, the tip of thefirst test probe 104 and the second test probe 105 are located indifferent regions within the fluid space 109 of the sample flow chamber106. The fluid space 109 within the chamber 106 may be of a first,smaller, cross-sectional area in a first region 114 of the fluid space109, and of a second, larger, cross-sectional area in a second region115 of the fluid space 109. An enlarging zone 116 may bridge the firstregion 114 and the second region 115. The tip of the first test probe104 may be substantially or fully in the first region 114 of the fluidspace 109, and the tip of the second test probe 105 may be substantiallyor fully in the second region 115 of the fluid space 109. In such aconfiguration, the flow rate around the tip of the first test probe 104will be different than the flow rate around the tip of the second testprobe 105. In such a configuration, the flow rate around the tip of thefirst test probe 104 will be lower than the flow rate around the tip ofthe second test probe 105. The differences between the flow rates of thefluid around the tips of the two probes presents a different environmentfor microbially influenced corrosion and for the formation of biofilm.

The test device 100 may have an inlet valve 112 adapted to shut off orallow fluid flow through the inlet tube 101. The outlet tube 102 maysimilarly have an outlet valve 113 to shut off or allow fluid flowthrough the outlet tube. An inspection cover 111 may allow for physicalinspection within the system, and the sample flow chamber 106.

In some embodiments of the present invention, as seen in FIG. 1B, asystem for bacteria detection using film formation promotion iscomprised of a test device 150 coupled to and adapted to route fluidfrom a fluid system, such as a water system, via an inlet tube 151. Theinlet tube 151 routes fluid into the chamber inlet 158 of a sample flowchamber 156. In some aspects, fluid from the fluid system is supplied tothe inlet tube 151 using a pump which pumps water from the main fluidsystem. In some aspects, fluid from the fluid system is supplied to theinlet tube 151 via gravity. In some aspects, the test device 150 isinverted to utilize gravity to create flow in the sample flow chamber156. In some aspects, a supply pump which pumps water from the mainfluid system into the inlet tube 151 is utilized to create flow in thesample flow chamber 156.

In some embodiments, a first probe apparatus 153 is removably insertedinto a first opening in the sample flow chamber 156. The probe apparatus153 may have a first test probe 154 coupled to a mounting unit adaptedto be inserted into the first opening in the sample flow chamber 156. Asecond probe apparatus 167 is removably inserted into a second openingin the sample flow chamber 156. The second probe apparatus 167 may havea second test probe 155 coupled to a mounting unit adapted to beinserted into the second opening in the sample flow chamber 156. In someembodiments, as seen in FIG. 1B, the tip of the first test probe 154 andthe second test probe 155 are located in different regions within thefluid space 159 of the sample flow chamber 156. The fluid space 159within the chamber 156 may be of a first, smaller, cross-sectional areain a first region near the fluid inlet 158 of the fluid space 109, andof a second, larger, cross-sectional area in a second region near theoutlet 157 of the fluid space 109. In such a configuration, the flowrate around the tip of the first test probe 104 will be different thanthe flow rate around the tip of the second test probe 105. In such aconfiguration, the flow rate around the tip of the first test probe 154will be lower than the flow rate around the tip of the second test probe155. The differences between the flow rates of the fluid around the tipsof the two probes presents a different environment for the formation ofbiofilm.

The test device 150 may have an inlet valve 162 adapted to shut off orallow fluid flow through the inlet tube 151. The outlet tube 152 maysimilarly have an outlet valve 163 to shut off or allow fluid flowthrough the outlet tube.

The first probe apparatus 153 may be coupled to an electronics unit 168with a cable 169. The second probe apparatus 167 may be coupled to theelectronics unit 168 with a cable 170. The electronics unit may beadapted to provide voltage differential to the two test probes whilemeasuring current flow. The electronics unit may be adapted to providedifferent voltage differentials, including reversing polarity of theprobes. The electronics unit may be adapted to implement instructionswhich direct the electronics to apply different voltage regimes overtime to the probes. The electronics unit may make intermittent orcontinuous measurement of current flow or other parameters.

In some embodiments of the present invention, as seen in FIG. 2, asample flow chamber 206 has an inlet 208 and an outlet 207. The sampleflow chamber has an increased cross-sectional flow area along the flowdirection 210. The first test probe 205 resides in a portion of thesample flow chamber 206 in a higher speed flow area relative to thesecond test probe 204. Both the first test probe 205 and the second testprobe 204 enter into the sample flow chamber 206 from a same end. Thedifferent flow rates seen by the two test probes result in differentconditions for the formation of biofilms at the two test probes. As seenin illustrative example in FIG. 4, the film growth 209 on the first testprobe 205 is different than the film growth 208 on the second test probe204. In some cases there may be a different amount of film growth on thetwo tips. In some cases there may be a different type of film grown onthe two test tips. In some cases, there may be similar film types on thetwo probes but in different quantities. In some cases, the differencesbetween the films may be significant enough that the films on the twotips exchange electrons, or other things, with the film on the othertip.

In some embodiments of the present invention, as seen in FIG. 3, asample flow chamber 226 has an inlet 228 and an outlet 227. The sampleflow chamber has an increased cross-sectional area along the flowdirection 230. The first test probe 225 resides in a portion of thesample flow chamber 226 in a higher speed flow area relative to thesecond test probe 224. The first test probe 225 enters into the sampleflow chamber 226 from a first end, and the second test probe 224 entersinto the sample flow chamber 226 from a second end. The different flowrates seen by the two test probes result in different conditions for theformation of biofilms at the two test probes. As seen in illustrativeexample in FIG. 5, the film growth 229 on the first test probe 225 isdifferent than the film growth 228 on the second test probe 224. In somecases there may be a different amount of film growth on the two tips. Insome cases there may be a different type of film grown on the two testtips. In some cases, there may be similar film types on the two probesbut in different quantities. In some cases, the differences between thefilms may be significant enough that the films on the two tips exchangeelectrons, or other things, with the film on the other tip.

In addition to the use of different flow rates to create differentgrowth environments and induce different amounts of microbe influencedcorrosion on the test probes, different temperatures may also be used tocreate different growth environments and induce different amounts ofmicrobe influenced corrosion on the test probes. In some embodiments, asseen FIG. 6, a sample flow chamber 236 has a constant cross-sectionalarea along the flow direction. A first test probe 235 is seen closer tothe inlet into the sample flow chamber and second test probe 234 is seencloser to the outlet from the sample flow chamber. Heaters 239 are usedto heat the water near the second test probe 234, and may also heat thesecond test probe 234. As seen, this creates different growthenvironments on the two test probes, which then results in an area ofmicrobially influenced corrosion 239 on the first test probe 235 that isdifferent than the area of microbially influenced corrosion 238 on thesecond test probe 234.

In another embodiment, as seen in FIG. 7, a sample flow chamber 246 hasa constant cross-sectional area along the flow direction. A first testprobe 245 is seen closer to the inlet into the sample flow chamber andsecond test probe 244 is seen closer to the outlet from the sample flowchamber. A heater 249 may be embedded within the second test probe 244.As seen, this creates different growth environments on the two testprobes, which then results in an area of microbially influencedcorrosion 249 on the first test probe 245 that is different than thearea of microbially influenced corrosion 248 on the second test probe244.

In some embodiments, the water temperature at the second probe is 20 Cwarmer than at the first probe. In some embodiments, the watertemperature is in the range of 10 C to 60 C warmer at the second probe.

In some embodiments of the present invention, as seen in FIG. 9, adouble chamber system 1000 flows fluid through a first sample flowchamber 1106, then heats the fluid in a fluid heater 1120, and thenflows fluid through a second sample flow chamber 1006. Fluid enters thedouble chamber system 1000 via an inlet 1101 which may be flowcontrolled by a valve 1112. In some aspects, a strainer 1123 may strainthe fluid prior to its delivery into the first sample flow chamber 1106.The first sample test chamber may have a first probe apparatus 1103which allows for ease in removing and replacing test probes into thefirst sample flow chamber 1106. The first probe apparatus 1103 may allowfor easy insertion of a first test probe 1004 and the second test probe1005. The sample flow chamber has an increased cross-sectional areaalong the flow direction. The first test probe 1004 resides in a portionof the sample flow chamber 1106 in a higher speed flow area relative tothe second test probe 1005. The different flow rates seen by the twotest probes result in different conditions for the formation of biofilmsat the two test probes.

The fluid exits the first sample flow chamber 1106 via an outlet 1121. Afluid sample heater 1120 heats the fluid prior to its entry into thesecond sample flow chamber 1006 via an inlet 1122. The second sampletest chamber may have a first probe apparatus 1003 which allows for easein removing and replacing test probes into the first sample flow chamber1006. The second probe apparatus 1003 may allow for easy insertion of afirst test probe 1104 and the second test probe 1105. The sample flowchamber has an increased cross-sectional area along the flow direction.The first test probe 1104 resides in a portion of the sample flowchamber 1006 in a higher speed flow area relative to the second testprobe 1105. The different flow rates seen by the two test probes resultin different conditions for the formation of biofilms at the two testprobes. In addition, the fluid temperature in the second sample flowchamber 1006 is warmer than in the first sample flow chamber 1106 as aresult of having been heated by the fluid sample heater 1120. Theenvironment for microbially enhanced corrosion is thus different in thesecond sample flow chamber 1006 than in the first sample flow chamber1106. This second growth environment allows for a second environment forfilm and corrosion promotion, allowing for an increased likelihood ofpromoting growth in one of the two sample flow chambers than may bepossible with a single growth environment.

In some embodiments, as seen in FIG. 8, the two test probes not onlyreside in areas within the sample flow chamber 256 which have differentflow rates but the two test probes are also at different temperatures.

In some embodiments, as seen if FIGS. 9 and 10, the sample flow chamberseach have two sets of test probes. In some embodiments, as seen in FIG.10, a sample flow chamber 276 with an inlet 282 and an outlet 283 with aheater 277 between the sets of test probes. The first test probes 278,279 are in a first environment wherein the fluid flow has not yet beenheated by the heater 277. The heater 277 heats the fluid so that thefluid flowing by the second test probes 280, 281 is warmer than it wasat the first test probes. Where mixed species biology may be present influid, their presence may be indicated between two or more sets ofsensor pairs where the fluid temperature is higher on one or more setsof sensor pairs in the array of sensor pairs. Where biology is notliving and not colonizing the sensors in the array, the effects of thedifference in temperature serve as a base line in the fluid beingmonitored. Where the differences in signals become different from thedetermined base-line, the presence of live mixed species biology may beindicated.

In some aspects, as seen in FIG. 11, a sample flow chamber 286 with aninlet 291 and an outlet 292 has an increase in cross-sectional flow areabetween the sets of test probes. The first test probes 287, 288 are inan area where the fluid flow velocity is higher than the fluid flowvelocity in the area of the second test probes 289, 290. The first testprobes 287, 288 are in a first environment wherein the fluid flow isquicker than further into the chamber. The fluid flow slows down in thearea of the second test probes 289, 290. FIG. 12 illustrates the filmgrowth 293, 294 on the first test probes 287, 288 and the film growth295, 296 on the second test probes 289, 290.

FIG. 13 illustrates an aspect of microbially influenced corrosion seenin systems according to embodiments of the present invention. In asystem using test probes, such as test probes made from mild steel,there will be general corrosion even in the absence of any livebacteria. Also, there will be microbially influenced corrosion whenthere are live bacteria. In a normal situation without differentiatedenvironments, bacteria may begin to influence corrosion without anysignificant likelihood that bacteria will colonize other than in randompatterns. However, by presenting differing environments on the two testprobe tips, either by having a different flow rate around each tip, or adifferent temperature at each tip, or both, it is very likely that thetype of colonization will be different. Because of this difference, itis likely that a net electron flow will occur between the bacteriallyinfluenced corrosion areas on the two tips. This may occur because thereis more oxidation occurring on one tip and more reduction occurring onthe other tip. The net electron flow is illustrated in FIG. 13.

In embodiments where there is a fluid pressure difference between thesensors, which will occur in constant flow systems with a difference inflow chamber cross-sectional area, oxygen as a compressible gas mayexpand when a fluid containing oxygen enters the area with lowerpressure. Where two biofilm communities are adjacent to each other, andwhere one is in an environment where oxygen is forming bubbles as itexpands under lower fluid pressure, different biofilm communities willestablish and exchange electrons in response to the difference in oxygenconcentrations in the fluid.

In some embodiments, as seen in FIG. 14, a system for bacteria detectionusing film formation promotion is comprised of a test device 1200 with asample flow chamber 1259. The sample flow chamber 1259 increases incross-sectional area along the flow direction between the chamber inlet1258 and the chamber outlet 1257. Fluid enters through an inlet 1251which may be controlled with a valve 1252. Fluid exits through an outlet1257. A test probe apparatus 1253 may be inserted into an opening in thesample flow chamber 1259. An electrical cable 1270 connects the testprobe apparatus 1253 to a system electronics 1268.

A series of test probes 1255 may include 6 sensor electrodes with metalsurfaces attractive to microbial corrosion. The fluid velocity decreasesalong a vertical axis across array of sensors due to fluid dynamicswithin the sample flow chamber. The differential flow creates differentmicro-environments which may promote differential microbial growth onthe different metal sensors. Using current readings as different voltagedifferentials are applied to the sensors, and with the use of switchingpolarity of the differential, as describe herein, allows for detectionof live mircro-organisms on a much quicker time scale than withconventional methods. In some embodiments, as seen in FIG. 15, a testsystem 1300 is similar to that of FIG. 14 with the sensor array 1355made up of protruding metal electrode rings.

In some embodiments, as seen in FIGS. 16A and 16B, a flow restrictionorifice 1400 may be used within the narrow entry section 1401 of thesample flow chamber. The use of the flow restriction orifice may allowan operator to tune the absolute and/or relative flow rate of theflowing liquid as they pass the first sensor tip and the second sensortip. The use of a flow restriction orifice may bring the flow rate downand also accentuate the difference in flow rate between the two sensortips.

An exemplary embodiment may be calibrated to operate with fluid flowvelocities between 0.5 feet per second and 10.0 feet per second in thesmall id section of the sample chamber. Over this range, the larger idsection will have 25 times less fluid flow velocity over a portion ofthe sensor element most extended into the larger id portion of thesample chamber. Under some applications, the fluid flow may be startedand stopped at time intervals between 1 to 23 hours on, in each 24 hourperiod. The fluid flow velocity may be as high as 20 feet per second inthe smaller id section of the sample chamber. In some aspects, not allof a sensor tip be in one constant velocity of fluid flow, similarly 100percent of the other sensor tip may not be in another and differentconstant velocity of fluid flow. Part of the sensor tips may have avariable fluid flow velocity over each. In such a circumstance theaverage fluid flow velocity over all of one sensor tip will still bedifferent than the average fluid flow velocity over all of the secondsensor tip.

A exemplary ratio of cross-sectional areas for flow in the samplechamber is: Flow Outlet side: 0.736310778 sq-in. to flow Inlet side:0.029351832 sq-in., the ratio of which is: 25 to 1. In some aspects, theratio will be in a range of 1.5:1 to 50:1. In some aspects, asignificant dynamic pressure drop may not take place until the ratio ismore than 20 to 1.

In a measurement method according to embodiments of the presentinvention, fluid flows into the sample flow chamber via an inlet. Insome embodiments this fluid is water diverted from a water system. Thefluid flows through the sample flow chamber and exits via an outlet.Within the sample flow chamber are two test probes subjected todifferent environments. In some aspects, the two test probes may havedifferent flow rates around their tips which has been induced by varyingthe cross-sectional flow area through the sample flow chamber. In someaspects, the two test probes are at different temperatures. In someaspects, both the flow rate and temperature differ between the two testprobe tips.

The two test tips are subjected to a voltage differential and thecurrent between the two test probe tips is monitored and recorded. Thepolarity of the voltage is then reversed and the current is also thenmonitored and recorded. In some aspects, each polarity is maintained forthe same amount of time between reversals. In some aspects the time is 3minutes. In some aspects the time between reversals is in the range of1-5 minutes. A change in current over time is expected even in theabsence of live microbes due to general corrosion.

The different environments on the two test probe tips result indifferent colonization types. The difference will be enough to result ina net electron flow between the two tips due just to the different typesof corrosion going on at the two test probe tips. This net current willrise to a level that can be seen in the current measurements takenacross the two tips. As the induced current due to the applied voltagereverses every few minutes, in one polarity direction the differentialcorrosion current will add to the induced current, and in the otherpolarity direction the differential corrosion current will subtract fromthe induced current. This difference in the absolute value of thecurrent indicates that film forming bacteria have begun to corrode thetips. This in turn indicates that there are indeed live film formingbacteria in the tested liquid sample.

The use of differing environments on the two test tips allows formeasurement of microbe influenced corrosion, thus detecting live filmforming bacteria much more quickly than prior methods. For example,detection can occur on the order of 4 hours after the live bacteria haveinfiltrated the system. In other aspects, detection may occur on theorder of 12 hours. The detection can occur before the main system hasfilmed up to the point of significant impact to the water system,allowing for possible treatment of the water with a biocide beforesignificant impact to the water system.

A description of the data displayed and the data provided as 4 to 20 MAoutput for interface to monitoring systems may be as described below. Toprovide a useful output in response to the electrical signatures presenton the sensor elements, changes in the electrical signature may bemeasured, displayed, and provided as an output to monitoring equipmentin a variety of ways.

In some applications, the lowest readings may not necessarily representthe presence of live mixed species bacteria, archaea and/or fungi. Thepresence of direct current stray current in electrically conductivefluid may be detected by any sensor elements in any conductive fluidbeing monitored. Therefore, in applications where continuous monitoringwill be established, a base line may be studied to account for thelowest readings observed. If the baseline is found to be an issue ofconcern, an investigation may be called for. Stray current of the directcurrent variety may continuously or intermittently affect many sensorsin electrically conductive fluids. ORP sensors, for example, are oftenaffected by stray current.

A suitable method to remove stray current from sensitive electricalsensor elements is to incorporate two sections of stainless steel pipesseveral feet upstream and downstream of the fluid conduit wheremonitoring is taking place. By grounding the 2 sections of stainlesssteel pipes, any stray current in the fluid will be removed from thefluid.

When mixed species biofilms become established on sensor elements, theybegin to support charge transfers of electrical energy through theirmetabolic processes, such as: Sulfate reduction; Sulfur oxidation; Ironor manganese oxidation; Iron reduction; Organic acid production; andCathodic depolarization through hydrogen utilization.

In stable fluids without stray current or live biology, very littledifference in potential between the like metal sensor elements in thefluid is expected. If we observe rapid and sudden changes in the data,this may indicate the presence of stray current from ungroundedequipment in electrical association with the conductive fluid we aremonitoring.

As biology begins to support charge transfer, the net electrical energydifference is usually a significant increase in the data displayed andmonitored. In methods according to embodiments of the present invention,the polarity of the charge transfer is encouraged and is not left torandom chance. The polarity of any stray dc current in the conductivefluid may add or subtract from the reading. A rapid and sudden shift inthe data up or down may be an indication of stray current. After theinfluence of stray current is eliminated, an increase over the course ofa day or more is a measurement of the net charge transfer.

From controlled testing with water drawn from ground wells, streams andponds, as the data on the display nears or exceeds a reading of 5.0units of measure, biofilm will always be found on the sensor elements.

When live biology first attach to the sensor elements there is nodetectable charge transfer from the first attachments. Over the courseof a number of hours or a few days, as the live biology colonize thesensor surfaces the charge transfer begins to be recognizable. In veryactive biology there is usually an increasing rate of detectable chargetransfer within days. After quite some time, such as several weeks ormonths the detectable electrical signature decreases and becomes maskedby the mature biofilm.

The detectable charge transfer increase more rapidly in response to thegrowth and maturity of the recently established biofilm. This oftenrepresents a non-linear increase in the reading on the display. Wherethe data ramps more rapidly after being low and somewhat flat, there isan indication that a biofilm is more strongly influencing the chargetransfer between the two sensor elements.

If the sensor elements are not replaced after a biofilm event and leftin place for months, the electrical signature will decrease over timeand may become useless. Continuous monitoring of the data and a reviewof the entire data from the time the sensor elements were newlyinstalled is recommended. Setting an alarm to detect an increase abovebaseline will also alert users to carefully inspect the fluid conditionsand/or sensor elements.

In some aspects, the control electronics 168 may include an alarm, whichmay be set to alarm at a level of current imbalance above a certainlevel. The control electronics reading may be an interpretation of thecurrent imbalance and may read out a different unit, which may be termeda biofilm unit.

As evident from the above description, a wide variety of embodiments maybe configured from the description given herein and additionaladvantages and modifications will readily occur to those skilled in theart. The invention in its broader aspects is, therefore, not limited tothe specific details and illustrative examples shown and described.Accordingly, departures from such details may be made without departingfrom the spirit or scope of the applicant's general invention.

What is claimed is:
 1. A method for the detection of live film formingmicrobes in liquid by stimulating differential growth, said methodcomprising the steps of: routing a liquid through an inlet of a sampleflow chamber; flowing a liquid through said sample flow chamber, saidsample flow chamber comprising a first test probe and a second testprobe, said liquid flowing first past said first test probe and thenpast said second probe; placing a voltage differential of a firstmagnitude and a first polarity across said first test probe and saidsecond test probe as a first voltage state; measuring the current flowbetween said first test probe and said second test probe in said firstvoltage state; placing a voltage differential of a first magnitude and areversed polarity of said first polarity across said first test probeand said second test probe as a second voltage state; and measuring thecurrent flow between said first test probe and said second test probe insaid second voltage state.
 2. The method of claim 1 further comprisingthe step of contrasting the current flow in said first voltage state andsaid second voltage state to detect the presence of microbiallyinfluenced corrosion.
 3. The method of claim 1 wherein the fluid flowvelocity around said first test probe is greater than the fluid flowvelocity around said second test probe.
 4. The method of claim 2 whereinthe fluid flow velocity around said first test probe is greater than thefluid flow velocity around said second test probe.
 5. The method ofclaim 3 wherein the ratio of the fluid flow velocity around said firsttest probe to the fluid flow velocity around said second test probe isin the range of 1.5:1 to 50:1.
 6. The method of claim 3 wherein theratio of the fluid flow velocity around said first test probe to thefluid flow velocity around said second test probe is greater than 20:1.7. The method of claim 4 wherein the ratio of the fluid flow velocityaround said first test probe to the fluid flow velocity around saidsecond test probe is greater than 20:1.
 8. The method of claim 1 whereinsaid sample flow chamber further comprises a heater adapted to heat thefluid after it has passed said first test probe and before it has passedsaid second test probe.
 9. The method of claim 2 wherein said sampleflow chamber further comprises a heater adapted to heat the fluid afterit has passed said first test probe and before it has passed said secondtest probe.
 10. The method of claim 3 wherein said sample flow chamberfurther comprises a heater adapted to heat the fluid after it has passedsaid first test probe and before it has passed said second test probe.11. The method of claim 8 wherein the fluid around said second testprobe is in the range of 10 degrees C. to 60 degrees warmer than thefluid around said first test probe.
 12. The method of claim 9 whereinthe fluid around said second test probe is in the range of 10 degrees C.to 60 degrees warmer than the fluid around said first test probe. 13.The method of claim 10 wherein the fluid around said second test probeis in the range of 10 degrees C. to 60 degrees warmer than the fluidaround said first test probe.
 14. The method of claim 1 wherein saidsecond test probe is heated in the range of 10 degrees C. to 60 degreeswarmer than said first test probe.
 15. The method of claim 2 whereinsaid second test probe is heated in the range of 10 degrees C. to 60degrees warmer than said first test probe.
 16. The method of claim 3wherein said second test probe is heated in the range of 10 degrees C.to 60 degrees warmer than said first test probe.
 17. The method of claim6 wherein said second test probe is heated in the range of 10 degrees C.to 60 degrees warmer than said first test probe.
 18. The method of claim1 wherein said second test probe is heated at least 20 degrees warmerthan said first test probe.
 19. The method of claim 2 wherein saidsecond test probe is heated at least 20 degrees warmer than said firsttest probe.
 20. The method of claim 6 wherein said second test probe isheated at least 20 degrees warmer than said first test probe.